Cartilage is a highly specialized tissue which functions to resist compression and to absorb shock. The purpose of this project is to understand molecular mechanisms by which genes for cartilage components are regulated and expressed during normal development and in disease states. The alteration of cartilage matrix protein are likely to be associated with human diseases such as osteoporosis, osteoarthritis, and rheumatoid arthritis. We have cloned the entire human aggrecan core protein. The sequence consists of 2,316 amino acids and is about 75% identical to that of the rat aggrecan which we have previously cloned. We have characterized the type II collagen enhancer. The enhancer consists of several cis-acting elements which interact with different transcription factors. One of the transcriptional factors has been cloned, sequenced and found to be a family of zinc finger proteins. The genes for the rat and human link protein have been cloned and characterized. The promoter activity of the link protein has been examined by deletion analysis. A DNA fragment of the first intron has been found to increase the promoter activity of the link protein. The human aggrecan gene has been cloned and used to screen DNA from patients with cartilage diseases for genetic linkage studies. GRAMT=Z01DE00484 The purpose of this project is to understand the molecular basis of connective tissue components as well as their gene regulation in normal development and in disease state using transgenic mice as models. Transgenic mice created by injection of DNA into mouse embryos have been exploited for the elucidation of factors which determine tissue specificity of gene expression. Phenotypic changes due to expression of foreign gene center the control of tissue specific heterologous promoters have also been studied. Creation of transgenic animals which carry mutated exogenous genes as models for human genetic diseases of cartilage and basement membrane have been exploited. The recently developed technique of targeted homologous recombination makes it possible to suppress the function of a specific domain of a protein or eliminate the function of a protein. We are establishing embryonic stem (ES) cell lines carrying a gene targeted by homologous recombination of exogenous DNA construct and introducing recipient blastogenesis in an effort to obtain chimeric mice that contain the altered genetic information in their germ line.